Gene Targeting in Human Pluripotent Stem Cells
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Targeted homologous recombination (HR) is an essential tool in stem cell biology. It can be used to study gene function and is a highly developed technology in the mouse where precise genetic modifications are introduced into the genome via HR in mouse embryonic stem cells (mESCs). However, gene targeting has not been widely applied to the study of human pluripotent stem cells (hPSCs) due to its relatively low efficiency in human cell lines. To overcome this technical hurdle, we have developed and established a protocol that allows efficient gene targeting in hPSC lines. This chapter provides a detailed protocol for efficiently performing gene targeting in hPSCs by electroporation. The protocol describes methods for cell preparation, antibiotic selection, and excision of the selectable marker following gene targeting. While we can only target one allele at a time, HR covers a broad range of important applications including making knock-in reporter lines and knock-in lineage tracers, generating disease models that are caused by dominant mutants, repair of patient-derived induced PSCs that only involve a single allele mutation, and knocking out genes that are located on the X chromosome in male lines. When targeting to both alleles is needed, such as generation of a knockout cell line, the cells can be electroporated twice with targeting vectors designed to target each of the alleles. This protocol will find broad applications in generating lineage-specific reporter lines and point mutations in genetic repair in disease models using hPSCs.
- Irion, S., H. Luche, P. Gadue, H. J. Fehling, M. Kennedy and G. Keller (2007) Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol. 25, 1477–82. CrossRef
- Zwaka, T. P. and J. A. Thomson (2003) Homologous recombination in human embryonic stem cells. Nat Biotechnol. 21, 319–21. CrossRef
- Ruby, K. M. and B. Zheng (2009) Gene targeting in a HUES line of human embryonic stem cells via electroporation. Stem Cells. 27, 1496–506. CrossRef
- Bu, L., X. Jiang, S. Martin-Puig, L. Caron, S. Zhu, Y. Shao, et al. (2009) Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 460, 113–7.
- Zou, J., M. L. Maeder, P. Mali, S. M. Pruett-Miller, S. Thibodeau-Beganny, B. K. Chou, et al. (2009) Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell. 5, 97–110.
- Sedivy, J. M., B. Vogelstein, H. L. Liber, E. A. Hendrickson and A. Rosmarin (1999) Gene Targeting in Human Cells Without Isogenic DNA. Science. 283, 9. CrossRef
- Xue, H., S. Wu, S. T. Papadeas, S. Spusta, A. M. Swistowska, C. C. MacArthur, et al. (2009) A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells. Stem Cells. 27,1836–46.
- Davis, R. P., E. S. Ng, M. Costa, A. K. Mossman, K. Sourris, A. G. Elefanty, et al. (2008) Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood. 111, 1876–84.
- Wu, S., G. Ying, Q. Wu and M. R. Capecchi (2008) A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat Protoc. 3, 1056–76.
- Datsenko, K. A. and B. L. Wanner (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 97, 6640–5.
- Lee, E. C., D. Yu, J. Martinez de Velasco, L. Tessarollo, D. A. Swing, D. L. Court, et al. (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics. 73, 56–65. CrossRef
- Zhang, Y., F. Buchholz, J. P. Muyrers and A. F. Stewart (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 20, 123–8. CrossRef
- Zhang, Y., J. P. Muyrers, G. Testa and A. F. Stewart (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol. 18, 1314–7. CrossRef
- Lombardo, A., P. Genovese, C. M. Beausejour, S. Colleoni, Y. L. Lee, K. A. Kim, et al. (2007) Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 25, 1298–306. CrossRef
- Liu, G. H., K. Suzuki, J. Qu, I. Sancho-Martinez, F. Yi, M. Li, et al. (2011) Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell. 8, 688–94. CrossRef
- Gene Targeting in Human Pluripotent Stem Cells
- Book Title
- Human Pluripotent Stem Cells
- Book Subtitle
- Methods and Protocols
- pp 355-367
- Print ISBN
- Online ISBN
- Series Title
- Methods in Molecular Biology
- Series Volume
- Series Subtitle
- Methods and Protocols
- Series ISSN
- Humana Press
- Copyright Holder
- Springer Science+Business Media, LLC
- Additional Links
- genetic engineering
- reporter cell line
- homologous recombination
- gene targeting
- Industry Sectors
- Editor Affiliations
- ID1. Research Institute, CHOC Human Neural Stem Cell Resource, Children's Hospital of Orange County
- ID2. Beckman Research Institute, Center for Applied Technology Developmen, City of Hope
- Author Affiliations
- 1. Department of Reproductive Medicine, UCSD Medical Center, University of California, San Diego, San Diego, CA, USA
- 2. Primary and Stem Cell Systems, Research and Development, Life Technologies, Frederick, MD, USA
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